Nicole M. Nichols

A high-throughput assay for the comprehensive profiling of DNA ligase fidelity

DNA ligases have broad application in molecular biology, from traditional cloning methods to modern synthetic biology and molecular diagnostics protocols. Ligation-based detection of polynucleotide sequences can be achieved by the ligation of probe oligonucleotides when annealed to a complementary target sequence. In order to achieve a high sensitivity and low background, the ligase must efficiently join correctly base-paired substrates, while discriminating against the ligation of substrates containing even one mismatched base pair. In the current study, we report the use of capillary electrophoresis to rapidly generate mismatch fidelity profiles that interrogate all 256 possible base-pair combinations at a ligation junction in a single experiment. Rapid screening of ligase fidelity in a 96-well plate format has allowed the study of ligase fidelity in unprecedented depth. As an example of this new method, herein we report the ligation fidelity of Thermus thermophilus DNA ligase at a range of temperatures, buffer pH and monovalent cation strength. This screen allows the selection of reaction conditions that maximize fidelity without sacrificing activity, while generating a profile of specific mismatches that ligate detectably under each set of conditions.

DNA Cloning and Engineering by Uracil Excision

This unit describes a simple and efficient DNA engineering method that combines nucleotide sequence alteration, multiple PCR fragment assembly, and directional cloning. PCR primers contain a single deoxyuracil residue (dU), and can be designed to accommodate nucleotide substitutions, insertions, and/or deletions. The primers are then used to amplify DNA in discrete fragments that incorporate a dU at each end. Excision of deoxyuracils results in PCR fragments flanked by unique, overlapping, single‐stranded extensions that allow the seamless and directional assembly of customized DNA molecules into a linearized vector. In this way, multi‐fragment assemblies, as well as various mutagenic changes, can all be accomplished in a single‐format experiment. Two basic protocols on the methods of uracil excision‐based engineering are presented, and special attention is given to primer design. The use of a commercially available cloning vector and the preparation of custom vectors are also presented. Curr. Protoc. Mol. Biol. 86:3.21.1‐3.21.16.

Template-independent DNA polymerases.

Terminal deoxynucleotidyl transferase, purified from calf thymus, catalyzes the incorporation of deoxynucleotides to the 3′‐hydroxyl termini of DNA accompanied by the release of inorganic phosphate. Reaction conditions are described in this unit in addition to some applications, including cloning DNA fragments, labeling the 3′ termini of DNA with 32P, incorporating nonradioactive tags onto the 3′ termini of DNA fragments, and synthesizing model polydeoxynucleotide homopolymers.

DNA Repair Enzymes

In vivo DNA damage impacts the genetic stability of an organism; therefore, multiple pathways utilizing a large number of enzymes have evolved to repair DNA damage. This unit focuses on enzymes involved in base excision repair (BER). The BER enzymes possessing N‐glycosylase activity can find and remove a wide variety of damaged bases in a sea of normal bases. The combination of unique substrate specificity, accuracy, and robust in vitro activity of many of these enzymes has led to their use in various experimental techniques, including site‐specific DNA cleavage. The enzymes described in this unit are active on many substrates including oxidized purines and pyrimidines, alkylated bases, abasic sites, pyrimidine dimers, deaminated cytosines, and deaminated adenines. Curr. Protoc. Mol. Biol. 84:3.9.1‐3.9.12.

DNA‐Dependent DNA Polymerases

This unit presents characteristics and reaction conditions of the DNA‐dependent DNA polymerases, including E. coli DNA polymerase I and its Klenow fragment, T4 DNA polymerase, native and modified T7 DNA polymerase, phi29 DNA polymerase, Bst DNA polymerase, and Taq DNA polymerase. The unit also provides overviews of other classes of thermophilic DNA polymerases used in PCR applications (described fully in UNIT 15.1), and the rapidly expanding class of lesion‐bypass DNA polymerases that play a role in DNA damage repair. Curr. Protoc. Mol. Biol. 84:3.5.1‐3.5.19.

UNIT 3.8 RNA Polymerases

This unit describes DNA‐dependent, RNA‐dependent, and template‐independent RNA polymerases. DNA‐dependent RNA polymerases include the related bacteriophage T7, T3, and SP6 polymerases, the most commonly used RNA polymerases for in vitro transcription reactions. Reaction conditions to produce preparative quantities of transcribed RNA and labeled RNA probes are covered, as are the major applications of these reactions. Limitations of the E. coli RNA polymerase for these applications are also presented. The properties of the phi6 RNA‐dependent RNA polymerase (RdRp) and its use in RNAi experiments are also introduced. Poly(A) polymerase, a template‐independent polymerase, catalyzes the incorporation of AMP residues onto the free 3′‐hydroxyl terminus of RNA, utilizing ATP as a precursor. Specific reaction conditions of poly(A) polymerase, as well as applications including RNA tailing and 3′ end labeling, are discussed. Curr. Protoc. Mol. Biol. 84:3.8.1‐3.8.8.

UNIT 3.13 Ribonucleases

Ribonucleases (RNases) with different sequence or structural specificities are used for a variety of analytical purposes, including RNA sequencing, mapping, and quantitation. The development of RNase protection assays, structural determination assays, and the production of small interfering RNAs (siRNA) employed in RNA interference (RNAi) experiments has depended on the unique substrate specificities of commercially available RNases, including RNases A, I, T1, V1, HI, III, and Dicer. One very common application for high purity RNase A is also presented in this unit and involves hydrolyzing RNA that contaminates DNA preparations. RNase HII and the placental RNase inhibitor are also discussed. Curr. Protoc. Mol. Biol. 84:3.13.1‐3.13.8.

Author Correction: Successful amplification of DNA aboard the International Space Station

A correction to this article has been published and is linked from the HTML version of this article.

Successful amplification of DNA aboard the International Space Station

As the range and duration of human ventures into space increase, it becomes imperative that we understand the effects of the cosmic environment on astronaut health. Molecular technologies now widely used in research and medicine will need to become available in space to ensure appropriate care of astronauts. The polymerase chain reaction (PCR) is the gold standard for DNA analysis, yet its potential for use on-orbit remains under-explored. We describe DNA amplification aboard the International Space Station (ISS) through the use of a miniaturized miniPCR system. Target sequences in plasmid, zebrafish genomic DNA, and bisulfite-treated DNA were successfully amplified under a variety of conditions. Methylation-specific primers differentially amplified bisulfite-treated samples as would be expected under standard laboratory conditions. Our findings establish proof of concept for targeted detection of DNA sequences during spaceflight and lay a foundation for future uses ranging from environmental monitoring to on-orbit diagnostics.

Abstract 3620: Enhancing clinical utility of NGS with reduced bias, low DNA input, library construction

Early detection and diagnosis of cancer substantially increases the likelihood for successful treatment. Tools that aid in detecting and diagnosing cancer early, therefore, have the potential to greatly impact the clinical outcome for cancer patients. Next Generation Sequencing (NGS) has emerged as an important tool in this area. The technology is sensitive, fast and high throughput to allow sequencing of many samples at once. Unfortunately, many clinical samples go unanalyzed because they do not yield sufficient quantities of DNA to generate NGS libraries or the libraries generated require so many rounds of PCR amplification that they display extreme sequence bias. Bias not only hampers data analysis, but also increases costs by requiring excess sequencing to obtain sufficient coverage over all relevant genomic regions. To enable the increased use of NGS in the clinic and reduce the amount of sequence bias generated during library preparation, we have developed a PCR free library construction method that uses low quantities of DNA as input. As an initial test of the method, we generated PCR free libraries from 100ng, 50ng and 25ng of human genomic DNA. The libraries where pooled and sequenced on the Illumina NextSeq 500 instrument to approximately 10X coverage. All libraries, irrespective of input amount, showed minimal AT/GC bias and excellent coverage distributions, with most bases covered within 5X of the expected coverage depth. In addition, regions identified as difficult to sequence (Aird, D., et.al., 2011 and Ross, M. G., et.al., 2013) showed coverage at near expected levels for all libraries. This method can easily be adapted for use with extremely low DNA inputs by the introduction of a minimal number of PCR cycles. In fact, we have used this method to construct high quality NGS libraries with picogram quantities of DNA input. Standard library construction methods require DNA inputs of 2ug to 500ng when PCR amplification is omitted. This new method utilizes inputs as low as 25ng to generate high-quality PCR free libraries and picogram quantities when amplification is performed. We are currently investigating the possibility of reducing input levels further and exploring the limits of the method with low quality DNA samples. Interestingly, we have observed substantial sample loss during DNA shearing and reaction cleanup. Samples that do not require fragmentation, such as DNA isolated from plasma (cfDNA) and low quality FFPE DNA, may reduce the input requirements even further. Finally, this new method utilizes low sample and reagent volumes, possibly paving the way for its use in microfluidic devices. Citation Format: Lynne Apone, Pingfang Liu, Vaish Panchapakesa, Deyra Rodriguez, Karen Duggan, Krishnan Keerthana, Nicole Nichols, Yanxia Bei, Julie Menin, Brad Langhorst, Christine Sumner, Christine Chater, Joanna Bybee, Laurie Mazzola, Danielle Rivizzigno, Fiona Stewart, Eileen Dimalanta, Theodore Davis. Enhancing clinical utility of NGS with reduced bias, low DNA input, library construction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3620.